Feed mass having a modified protein structure for carnivorous animals

ABSTRACT

A method for producing feed for forming into pellets, the pellets produced by the method to be used to feed carnivorous animals. The addition of the enzyme transglutaminase to a feed mass specifically intended for carnivorous fish will catalyze a reaction between the amino acids glutamine and lysine which form part of the protein chains and the raw material of the proteins of the feed, such that a covalent chemical bond is formed between them, which results in shape permanence in the formed, dried, finished pellets, such that the finished pellets do not lose their shape before time of use.

This application is a divisional of U.S. application Ser. No.09/673,003, filed Oct. 6, 2000, now U.S. Pat. No. 6,399,117, which wasthe National Stage of International Application No. PCT/NO99/00102,filed Mar. 25, 1999.

BACKGROUND OF THE INVENTION

The present invention relates to a method for the modification ofprotein structure in finish shaped feed pellets, balls or the like,among other reasons in order to contribute to strengthening thepermanence of the pellet shape in granular feeds of this sort. Theinvention also relates to feedstuff manufactured according to thismethod for the formation of a shape-permanent feed in pellet form.

Many types of animals in breeding, e.g. salmon and trout, arecarnivorous (meat-eating). Their natural food consists of insects (forsalmon and trout in the freshwater phase), fish and crustaceans. Forother carnivorous farmed animals, such as mink and foxes, the feed mayalso contain the mammalian flesh, for example fresh slaughterhousewaste. Insects and crustaceans have an exoskeleton which i.e. consistsof chitin. Chitin is a linear polysaccharide of N-acetyl-D-glucosaminelinked by β1→4 bonds. Other structural carbohydrates such as cellulose(β[1→4]D-glycose) and alginate (D-mannuronic acid) are not found inthese prey animals, nor do they contain starch (á[1>4]D-glycose) asenergy stores. Nevertheless, salmon, trout and other carnivorous fishand animals have enzymes (for example, amylase) that are capable ofbreaking down starch in the gut and making it digestible, but they maybe less efficient in this respect than herbivorous (plant-eating) fishand animals.

In feeds intended for carnivorous fish it is usual to add between 8% and25% carbohydrates, for example in the form of wheat or maize, as abinding agent. After pressing, but especially after extrusion, thestarch in these carbohydrates will form a matrix or base mass whichgives the pellets mechanical strength and shape permanance so that theshape of the pellets can be maintained after drying, further processing,storage and transport.

Carbohydrates are utilised in metabolism as a source of energy. Theenergy density of carbohydrates is lower than that of protein and fat(17.6; 23.9 and 39.8 MJ/kg respectively). The digestibility ofcarbohydrates is also lower in carnivorous fish, and declines as theproportion of complex carbohydrates in the feed (above 10%) increases.Experiments have shown that salmonids have no metabolic need forcarbohydrates. If fat replaces carbohydrates as an energy source, acarbohydrate-free fish feed of this sort will contain more energy perunit weight, as long as the relative proportions of the other componentsare held constant.

In order to give feed pellets shape permanence and mechanical strength,as mentioned above, it is known to add a binding agent in the form of8%-25% carbohydrates, for example wheat and/or maize. After pressing orextrusion in the feedstuff material there will be established a starchmatrix of the desired strength.

Other techniques in connection with the forming of feed into pelletsballs or the like, have also been described. According to U.S. Pat. No.4,935,250, for example, a gel or mass of alginate is also producedduring the forming.

The patent literature includes descriptions of feeds and feed mixturesin which the mass consists of gelatine or caseinate. See, for example,British Patent No. 2,217,175.

There are also feeds in which the binding characteristics produced bythe coagulation of native proteins are exploited; see NO 179 731.

Small feed particles can be produced with the aid of an agglomerationtechnique, which are based on the principle of aggregating extremelysmall particles into larger particles. This process does not utilizecarbohydrates as a binding agent. The feed components are bound togetherthrough various forms of contact bonds between the solid particles inthe feed. The different forms of contact bonds can vary from hydrogenbonds, adhesion and cohesion to capillary forces. New covalent bonds arenot created in this process. This is an obvious disadvantage for themaintenance of the feed pellet's form and strength, because covalentbonds are stronger than other chemical bonds.

A serious disadvantage of agglomerated feeds is thus that the bonds areweak, and given the lack of a continuous matrix such pellets are friableand fragile. The agglomeration technique cannot be utilised to produceparticles of feed in pellet form with a diameter larger than about2.5-3.0 mm.

In order to be able to produce larger feedparticles/fragments/pellets/balls, etc., we must abandon theagglomeration technique without addition of carbohydrate and againreturn to carbohydrate as a binding agent. In low concentrations,complex carbohydrates such as starch are digested by salmonids, forexample, but if their concentration exceeds 10% the digestibility of thecarbohydrate fraction decreases (Aksnes A., 1995. Growth, feedefficiency and slaughter quality of salmon, Salmo salar L., given feedswith different ratios of carbohydrate and protein. AquacultureNutrition, 1:241-248).

GENERAL DESCRIPTION OF THE INVENTION

The energy content of the carbohydrate fraction may be replaced by fatwithin the feed recipe. This will result in greater freedom with respectto varying the relative proportions of fat, protein and micronutrientssince the carbohydrates make up the remainder of the feed recipe. Such afeed will be richer in energy than an equivalent feed containingcarbohydrates, and a reduction in the feed conversion ratio, defined asthe quantity of feed consumed to produce one kilo of fish biomass, willbe obtained.

In accordance with the present invention one has aimed at showing amethod of modifying the protein structure of feeds whose nutrientcomposition closely resembles the natural choice of foods of carnivorousfish and animals. According to the invention, favourable binding isobtained in feeds—without carbohydrates—resulting in shape permanence inpellets and similar forms of feedstuff, and in such a way that themaximum particle size/pellet diameter can be increased in the case ofagglomerated feed while maintaining the shape of the feed. The inventionalso aims to increase the energy density of all types of feed,particularly fish feeds.

The above-identified objective is reached by proceeding as describedherein. A feedstuff that has been treated as described in accordancewith this method and which is intended for the forming of pellets or thelike, and properties relating thereto are identified herein. Feedpellets made of such a feedstuff, whose protein structure has beenmodified by the method, are also disclosed herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The method and product formed by the method thus consists mainly of afeedstuff, or alternatively of one or more of the components of which itconsists, for forming pellets, alternatively of coating the surface ofthe formed pellets, in order to add an enzyme (preferablytransglutaminase) that function as a catalyst for the formation ofcovalent bonds between the amino acids which make up the protein chainsin protein raw materials containing proteins in native or denaturedform. The raw materials of the protein in the feed mixture include thenatural amino acids glutamine and lysine in the protein chains. Theadded enzyme will act as a catalyst and catalyze the formation ofcovalent (å-(γ-Glu)Lys) bonds between the amino acids glutamine andlysine in the protein raw ingredients in the feed.

Through the adoption of reaction temperature and reaction time thisenzymatic reaction will form a matrix or basic mass of protein rawmaterials which will exhibit adequate strength to give feed pellets aconstant and lasting shape.

In agglomerated feed this enzymatic reaction will lead to covalenttransverse bonds (cross-bonds) between the proteins. This will give theagglomerate increased strength by introducing the strongest type ofchemical bond in addition to the other three types of chemical bondswhich provide the feed product with firmness and strength.

In pressed or extruded feeds, the formation of covalent (å-(γ-Glu)Lys)bonds that lead to the formation of a protein matrix will be able topartially or wholly replace the addition of carbohydrates. This makes itpossible to remove carbohydrates from the recipe for the feed, or toreduce the proportion of carbohydrates respectively. Carbohydrates playvirtually no part in the natural diet of carnivorous fish and animals,and the total energy density of feed pellets can be increased becauseboth protein and fat are richer in energy than carbohydrates. Accordingto the present invention, an animal feed in the form of pellets willrequire no other binding agents such as gelling agents in the form ofgelatine, for example.

The above reaction formula, in which the enzyme transglutaminase acts asa catalyst, shows the creation of å-(γ-Glutamyl) lysine bond between theamino acids glutamine and lysine.

Transglutaminase is defined as enzymes which are classified asprotein-glutamine γ-glutamyltransferase (EC 2.3.2.13; InternationalUnion of Biochemistry and Molecular Biology, Nomenclature Committee).Transglutaminase may occur in a pure form or as a distinct premix withsuitable filler and transglutaminase in adjusted concentration.Transglutaninase can be added to the other raw ingredients of the feedin the form of a powder, in solution or in suspension.

The term “protein raw materials” refers to raw ingredients that containprotein in either native or denatured form.

Examples include fish meal, stickwater, stickwater concentrate, bloodmeal, feather meal, bone and horn meal, wheat gluten, maize gluten, soyameal and rapeseed meal. These are only illustrative examples, and do notexclude the use of other raw ingredients in feeds as partial substitutesfor or additions to one or more of the said raw ingredients.

The term “feed pellets” refers to particles or fragments, preferablyround, which are formed by means of a special process, such that theyare of a size and shape that makes them suitable as feed, particularlyfor carnivorous fish species such as farmed salmon, cod, halibut, seaperch and sea bream.

The activity of the enzyme (transglutaminase) declines at temperatureshigher than 50° C., and it is deactivated at temperatures beyond 65° C.The formation of a protein matrix as described here can thus take placeunder process heat conditions in which the process temperature variesbetween 0 and 60° C. The shortest process time is obtained at around 50°C., since the process time is lengthened at temperatures above and belowthis temperature.

Transglutaminase is added to the other ingredients of the feed beforethese are formed into pellets by a suitable method, since the enzyme canbe added as a solution or as a suspension in a suitable liquid, forexample water, or blended as a dry ingredient before liquid is added tothe mixture. In addition to transglutaminase, an aqueous solution maycontain pure water, stickwater or stickwater concentrate, or anotherprotein-rich liquid, for example a non-limiting gelatine solution.

Transglutaminase can also be added to the surface of preformed pelletsby a suitable method, in that the enzyme can be added as dissolved insteam, or as a solution, suspension or wash in a suitable liquid such aswater.

The fat content of the feed may, according to a non-limiting preferredexample, consist of fish-oil, which can be added to the feed eitherbefore or after the feed is formed into pellets, or after the pelletshave been dried. After the feed has been formed into pellets, thepellets are maintained at a temperature of between 0 and 60° C., so thatthe transglutaminase enzyme has time to catalyse the å-(γ-Glutamyl)lysine bonds that are desired.

The reaction time is adapted to the reaction temperature.

Finally, the formed pellets are dried to the desired watercontent/degree of dryness in a suitable dehydration unit, such as adrying cabinet.

The transglutaminase product used in the following examples (from themanufacturer Ajinomoto in Japan) consists of 60% sodium caseinate, 39.5%maltodextrin and 0.5% transglutaminase. A meat grinder with a die sizeof around 6.5 mm can be used to form the feed into pellets. Theapparatus may also comprise an incubator cabinet and a drying cabinet(working temperature about 80-90° C.). In a wear test, a rotation rateof 500 rpm was employed without the use of metal balls.

EXAMPLE 1

500 g fish meal (Norwegian LT meal) and 500 ml water were mixed toformation of a dough which was then formed into pellets with the aid ofa meat grinder. The formed pellets were dried for about 35 minutes. 300g pellets were used for the wear test.

25 g transglutaminase product containing 125 mg transglutaminase and 15g sodium caseinate was stirred into 500 ml water at about 40° C. Thesolution was added to 500 g fish meal (Norwegian LT meal), mixed to adough and formed into pellets with the aid of a meat grinder. Thesepellets were incubated in the said incubator cabinet for about 60minutes at about 40° C., and thereafter dried for about 50 minutes. 300g pellets were used for the wear test.

Result

Composition of fish meal protein 72.5% fat 8.7% ash 11.8% water 8.5%Water content meal + water 3.3% after drying meal + sodium 3.6%caseinate + water + transglutaminase Wear test, remaining meal + water1.2% pellets meal + sodium 86.3% caseinate + water + transglutaminase

The example shows that blending around 125 ppm transglutaminase into amixture of fish meal, sodium caseinate and water produced pellets withconsiderably greater firmness and strength than pellets formed from afeed mass (fish meal+water) without this enzyme additive.

The enzyme transglutaminase in powder form can be blended with one ormore of the other dry ingredients of the feed before water is added inthe form of liquid, for example pure water, stickwater, stickwaterconcentrate, other protein-enriched liquid or water vapour.

Drying may take place immediately after forming into pellets, as long ascare is taken to ensure that the temperature does not rise above 60° C.,thus giving the enzyme sufficient time to act to create a protein matrixbefore the water activity becomes so low that the enzyme will no longeract as a catalyst.

Transglutaminase produced by temperature-tolerant bacteria will be ableto act at temperatures higher than 60° C.

Production conditions can thus be above 60° C. if thermo-stabletransglutaminase is employed.

EXAMPLE 2

In this case another source of protein (soya) was used, whose realprotein level is about 20% lower than that of fish meal. The feedstuffwas prepared largely as indicated in Example 1.

500 g soya meal (Hamlet) and 700 ml water were mixed to the formation ofa dough, which was formed into pellets and dried for about 40 minutes.As in example 1, a wear test was carried out. After the test had beencompleted, the remaining whole pellets were subjected to a new weartest, this time with four metal balls and at 150 revolutions. 25 gtransglutaminase product containing 125 mg transglutaminase and 15 gsodium caseinate was stirred into 700 ml water at about 40° C. Thesolution was added to 500 g soya meal flour, blended and formed asindicated in example 1, incubated at 40° C. for 60 minutes and dried forabout 60 minutes.

Result

Composition of soya meal protein 56.5% fat 1.0% ash 7.1% water 7.9%Water content meal + water 10.0% after drying meal + sodium 7.2%caseinate + water + transglutaminase Wear test without balls, meal +water 85.6% remaining pellets meal + sodium 95.8% caseinate + water +transglutaminase Wear test without balls, meal + water 35.5% remainingpellets meal + sodium 72.0% caseinate + water + transglutaminase

The invention also comprises other enzymes that catalyse the formationof covalent bonds between amino acids that form part of the proteinchains in the raw materials of the protein in the feed.

Such an enzyme can, for example, catalyse an equivalent reaction betweenasparagine and lysine (å-(β-aspartyl) lysine bond) (transasparaginase).

Other examples are the enzyme protein disulphide isomerase (EC 5.3.4.1),which catalyses the rearrangement of cystine and the enzymelipoxygenase.

A method for making feed for carnivorous animals, especially feed forfarmed fish, including modifying protein structure of pre-shaped feedpellets, spheres, and similar feed particles or pieces, in order toachieve shape permanence, strength and firmness of said feed pellets andthe like, wherein said properties are maintained subsequent to apossible incubation, drying, further processing, storage and transport,and wherein is used a catalyzing enzyme such transglutaminase,characterized in that said enzyme catalyzing the formation of covalentbonds between amino acids included in the protein chains of the proteinraw material containing proteins in native or denatured form and that,to a substantial degree, constitutes structureless processed rawmaterials such as stickwater, stickwater concentrate, and structurelessdry processed raw materials (powders) such as fish meal, blood meal,feather meal, or bone meal, wheat gluten, maize gluten, soy meal, canolameal, casein, sodium caseinate, gelatin or collagen, is mixed to theready-mixed feed mass or, alternatively, to one or more of itsingredients prior to mixing, in order to build a protein network of saidstructureless raw materials, whereupon the feed mass to which thecatalyst is added in the form of said enzyme is formed into feed pelletsor the like. Alternatively, that said enzyme is applied to the surfaceof pre-shaped feed pellets, which are incubated at a temperature and fora period adjusted according to the kinetic properties of the enzyme,such that covalent bonds are established between said amino acids, andthat said feed pellets or the like are further processed, conservationbeing effected through drying, and that, subsequent to the pelletshaping process, a reaction temperature is established in the range of0-60° C., preferably 10-50° C., and wherein pre-shaped feed pellets,spheres and similar feed particles exhibit the desired shapeperformance, said protein network having been built up in such a waythat it does not only form gel, but such that increased mechanicalstrength is established and maintained subsequent to the drying.

The method further wherein a catalyzing enzyme is used, such astransglutaminase, transasparaginase, protein disulphide isomerase and/orlipoxygenase, characterized in that said enzyme(s) is (are) added to thefeed mass in an amount corresponding to at least 10 ppm of the (dry)weight of the feed mass and that, subsequent to the pellet shapingprocess, the reaction temperature is established in the range of 0-60°C., preferably 10-50° C., (does not apply for thermostabiletransglutaminase that may function as a catalyst at temperatures greaterthan 60° C.), whereupon feed pellets are incubated and dried (e.g. in 60and 50 minutes, respectively, dependent upon the temperature), so thatthe catalyzing property of the enzyme ceases when said feed pellets arepre-dried.

The method further characterized in that the enzyme transglutaminase areadded in powder form to dry feed mass or, alternatively, to one or moreof its ingredients prior to mixing, the transglutaminase powder beingmixed into the same at the stage prior to the mixture of water into themixture in the form of a liquid, such as pure water, stickwater,stickwater concentrate, other protein containing liquid and/or watervapor.

The method further characterized in that to a blending liquid for thefeed mass (which has approximately the same weight as thelast-mentioned) is added about 20 ppm or more transglutaminase, referredto the weight of the blending liquid, said transglutaminase beingstirred and agitated into the blending liquid to form thereon an aqueoussolution or suspension, the solution or suspension being mixed with adry feed mass of approximately the same weight as the blending liquid.

The method further characterized in that the enzyme transglutaminase, isin a condition wherein it is dissolved in vapor, forming a solution,suspension or washing in a suitable liquid, such as water, is applied tothe surface of pre-shaped feed pellets and that, subsequently, to thepellet shaping process, a reaction temperature is established within therange of 0-60° C., preferably 10-50° C., (not including thermostabiletransglutaminase which may function as a catalyst at temperaturesgreater than 60° C.), whereupon feed pellets are incubated and dried(e.g. for 60 and 50 minutes, respectively, dependent upon thetemperature), so that the catalyzing property enzyme ceases when saidfeed pellets are pre-dried.

A feed mass intended for the formation of feed particles (pellets), forcarnivorous animals, especially farmed fish, prepared, made andprocessed in accordance with the method as set forth in any of thepreceding disclosure, and where any catalyzing enzyme, particularlytransglutaminase, has been added to said feed mass, characterized inthat said catalyzing enzyme, such as transglutaminase, within said feedmass has catalyzed a reaction between amino acids, especially glutamineand lysine, included in the protein chains of the protein raw materialof the feed mass, said raw material containing proteins and native ordenatured form and which, to a substantial degree, constitutesstructureless processed raw materials, such as stickwater, stickwaterconcentrate, and structureless dry processed raw materials (powders),such as fish meal, blood meal, feather meal or bone meal, wheat gluten,maize gluten, soya meal, canola meal, casein, sodium caseinate, gelatin,or collagen.

The feed mass further characterized in that the feed mass also containsmicronutrients.

The feed mass further characterized in that the feed mass is conservedexclusively through drying.

The use of enzyme transglutaminase as a catalyst for a protein and aminoacid containing feed mass to be shaped into pellets for farm fish, withthe purpose of catalyzing a reaction resulting in the formation ofcovalent bonds between amino acids included in the protein chains of theraw materials of said feed mass, and said raw materials thereofcontaining proteins in native or denatured form.”

What is claimed is:
 1. A feed for carnivorous animals comprising: A. aprotein raw material having a protein structure and a catalyzing enzyme,said catalyzing enzyme forming covalent bonds between amino acids ofproteins of the protein raw material, the protein raw materialcomprising proteins in denatured form, the protein raw materialcomprising structureless processed raw materials being selected from thegroup consisting of stickwater, stickwater concentrate, andstructureless dry processed raw materials in the form of powders, or anycombination thereof, said powders being selected from the groupconsisting of fish meal, blood meal, feather meal, bone meal, wheatgluten, maize gluten, soy meal, canola meal, casein, sodium caseinate,gelatin, and collagen or any combination thereof, said catalyzing enzymebuilding a protein network for said structureless processed rawmaterials, wherein said protein raw material and said catalyzing enzymeare shaped into consumable feed pellets having shape permanence,strength, and firmness, said consumable feed pellets having a watercontent equal to or less than 20%.
 2. The feed according to claim 1,wherein said catalyzing enzyme is selected from the group consisting oftransglutaminase, transasparaginase, protein disulfide isomerase andlipoxygenase, or any combination thereof, said catalyzing enzymecomprising an amount of approximately at least 10 parts per million ofthe weight of the protein raw material.
 3. The feed according to claim2, wherein said catalyzing enzyme is transglutaminase, saidtransglutaminase comprising powder transglutaminase, wherein said powdertransglutaminase is combined to said protein raw material.
 4. A feed forcarnivorous animals comprising: A. a raw protein material, said rawprotein material having protein structure, said protein structurecomprising protein chains and protein in a denatured form, said proteinchains having covalent bonds and amino acids, said raw protein materialbeing structureless processed raw material selected from the groupconsisting of stickwater, stickwater concentrate, fish meal, blood meal,feather meal, bone meal, wheat gluten, maize gluten, soy meal, canolameal, casein, sodium caseinate, gelatin, and collagen and anycombination thereof; B. a catalyzing enzyme, said enzyme comprisingtransglutaminase, said catalyzing enzyme catalyzing the formation ofcovalent bonds between said amino acids, wherein said raw proteinmaterial and said catalyzing enzyme are formed into consumable feedpellets having enhanced shape permanence, strength, and firmness.
 5. Thefeed according to claim 4, wherein said catalyzing enzyme is selectedfrom the group consisting of transglutaminase, transasparaginase,protein disulfide isomerase, lipoxygenase, and any combination thereof,and further said catalyzing enzyme comprising an amount of at least 10parts per million as compared to the dry weight of the feed.
 6. The feedaccording to claim 5, wherein said catalyzing enzyme is transglutaminasein a powder form wherein said powder transglutaminase is combined tosaid structureless processed raw materials, said feed further comprisinga blending liquid, said blending liquid being selected from the groupconsisting of water, pure water, stickwater, stickwater concentrate,water vapor, and protein containing liquids or any combination thereof.7. The feed according to claim 5, wherein said blending liquid hasapproximately the same weight as the raw protein materials and furtherwherein about 20 parts per million of transglutaminase is combined tosaid blending liquid.
 8. The feed according to claim 4, wherein saidtransglutaminase is a solution.
 9. The feed according to claim 4,wherein said consumable feed pellets are substantially